Homer3 regulates the establishment of neutrophil polarity.

Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference-mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP3) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP3 and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.

Escherichia coli peptide binding protein OppA has a preference for positively charged peptides.

The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 Å) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 Å, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.

An immunological evaluation of type II diabetic patients with periodontal disease.

Peridontal disease is a frequent complication of diabetes, and diabetic subjects often exhibit decreased immune response with increased susceptibility to infection. We evaluated the possible relationship between immune response and periodontal disease in 40 type II diabetic patients, mean (+/- SD) age 59 +/- 8 years and mean disease duration 17 +/- 4 years, with good metabolic control (mean fasting plasma glucose, 10.5 +/- 3.8 mM/L, mean HbA1c 8.1 +/- 1.66%), and in 40 age and gender-matched controls. Interproximal alveolar bone loss (ABL), as the percentage of bone loss from the cement enamel junction (CEJ) to the apex, was measured with a modified Schei ruler at the deepest point on the mesial/distal surface of the teeth, except third molars, on a panoramic radiograph. Immunological evaluation involved study of NADPH neutrophil superoxide production, neutrophil chemotaxis, lymphocyte subpopulations, immunoglobulins and complement. Diabetic patients showed significant differences compared with controls regarding ABL (30.6 +/- 14.7% versus 17.6 +/- 4.3%; p < 0.0001) and the T-helper/T-suppressor ratio (2.3 +/- 1.0% versus 1.8 +/- 0.8%; p < 0.05). Other parameters of cell-mediated immunity and humoral immune response did not show any significant variations. No correlation between immunological and radiographic analysis parameters were found. Further studies are needed to verify the exact role played by immunological factors in type II diabetic patients with periodontal disease.

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria.

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.

Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria.

The 2.0 A crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals a fold similar to that of tyrosine hydroxylase. It provides the first structural view of where mutations occur and a rationale to explain molecular mechanisms of the enzymatic phenotypes in the autosomal recessive disorder phenylketoneuria.

Purification and light-scattering analysis of penicillin-binding protein 4 from Escherichia coli.

Penicillin binding protein 4 (PBP4) from Escherichia coli is a protein involved in the recycling and maturation of the bacterial cell wall and it is inhibited by beta-lactam antibiotics. PBP4 exhibits D-Ala-D-Ala-endopeptidase as well as D-Ala-D-Ala-carboxypeptidase activity. To provide a structural template for the design of new, more specific antibiotics we started X-ray crystallographic studies of penicillin binding protein 4 from Escherichia coli. PBP4 has been overexpressed in Escherichia coli as a His-tagged protein. A large-sclae purification scheme, yielding a very pure material, has been set up and crystallization experiments have been started. Dynamic light scattering experiments suggested that PBP4 exhibits aggregation behavior with a number of different precipitating agents and additives. Only by addition of EDTA, PEG 4000, and ammonium sulfate is the molecular mass about 110 kDa.

Purification, crystallisation and preliminary X-ray analysis of penicillin binding protein 4 from Escherichia coli, a protein related to class A beta-lactamases.

Crystals of the penicillin binding protein 4 (PBP4) from Escherichia coli have been obtained at 37 degrees C from liquid to liquid diffusion experiments in capillaries. PBP4 was dissolved in a 1.0 M ammonium sulphate solution, buffered at pH 7.2, to a concentration of 5 mg/ml, and was layered on top of a 1.6 to 2.2 M ammonium sulphate solution. Crystals appeared within four to six weeks. They belong to space group C222 with cell dimensions a = 68.5 A, b = 100.5 A and c = 137.0 A, and diffract to at least 2.8 A resolution. There is one molecule with a molecule mass of 49,568 Da in the asymmetric unit.

Variability within the Candida rugosa lipases family.

Several fungi secrete lipase isozymes differing in biochemical properties and in some cases in substrate specificity. In the yeast Candida rugosa, a family of related genes encodes for multiple lipase proteins, highly homologous in sequence but partially different in the regions interacting with the substrate molecule. Analysis of these substitutions performed on the basis of multiple alignments and using a 3-D model of the enzyme, allows identification of a restricted number of amino acids possibly involved in substrate specificity of Candida lipases.

Cloning and analysis of Candida cylindracea lipase sequences.

Lipases (Lip) hydrolyze triglycerides into fatty acids and glycerol. Lip produced by the yeast Candida cylindracea are encoded by multiple genomic sequences. We report the molecular cloning and characterization of three genes from this family. They encode putative mature 57-kDa proteins of 534 amino acids (aa). To date, five Lip-encoding genomic sequences from C. cylindracea have been characterized in our laboratory. The five deduced aa sequences share an overall homology of 80%. These sequences have been aligned with each other and with those of homologous enzymes, the Lip from the mould Geotrichum candidum and the acetylcholinesterase from Torpedo californica, whose three-dimensional structures have been solved by X-ray analysis. The C. cylindracea Lip appear to have a structural organization similar to that described for both enzymes.

Homology-derived three-dimensional structure prediction of Candida cylindracea lipase.

We propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed.

Cloning and nucleotide sequences of two lipase genes from Candida cylindracea.

Two lipase-encoding genes (LIP1 and LIP2) have been isolated from a SacI genomic library of the yeast Candida cylindracea and their nucleotide sequences have been determined. Comparison with the sequence of a cDNA ruled out the presence of introns in the two genes. Both ORFs encode for mature proteins of 534 residues with putative signal peptides of 15 and 14 amino acids, respectively. When compared with other lipase sequences, the two C. cylindracea lipases showed homology only with the Geotrichum candidum lipase, whereas they shared a significant similarity with several esterases.